We will propose a method to detect changes in intramolecular structure or intermolecular organization based on orientation imaging of fluorescent single molecules. We will develop robust instrumentation for polarized fluorescence imaging exhibiting the speed and sensitivity required to monitor 3D angular changes of individual fluorophores that are rigidly connected to proteins of interest. While developing the optical arrangement and required acquisition and processing algorithms, we will use the system to monitor the organization of septin molecules in a filamentous fungus, Ashbya gossypii and in budding yeast. These model systems are used because they are highly amenable to imaging, molecular genetic and biochemical manipulations and the septins in these cells are well characterized. The septins are a highly conserved component of the cytoskeleton that are critical for cytokinesis and intracellular compartmentalization. Important insights have been gained about the steady state organization of septins using polarized fluorescence imaging approaches but never at the single molecule level. As Aim 1 we will develop instrumentation and probe design for imaging the 3D orientation imaging of fluorescent single molecules. For Aim 2 we will analyze the mechanisms of septin assembly and reorganization in vitro and in living cells. For Aim 3, we will reveal the mechanisms of diffusion barrier function of septins in cellular membrane. In practice, we anticipate continuous back-and-forth transitions between those methods development and biological applications.